Over the last project period, we have achieved the goal of detecting the expression of many genes in a single cell using multiplexed probes to develop a barcode for eleven transcription sites for specific genes and their alleles. This single cell gene expression profiling method allows assessment of each cell's pattern of expression as well as allowing a population analysis of the regulation of these genes. The extensive data complexity that results from the gene-to-gene analyses indicates that this will be an informative approach to validate the gene expression patterns pertaining to cancer gene expression as well as to discover new correlations of genes with the cancer phenotype. The aim of this proposal is to apply this methodology we have developed to tissue samples in order to derive specific gene expression information, ultimately from patient specimens. The approach is to optimize this technology by developing reagents, protocols and imaging hardware and software to achieve a high signal of expressed genes and reduced background of tissue autofluorescence. Central to this development is the use of two new instruments, the Leica confocal microscope, and the VarispecTM liquid crystal tunable filter, which allow a complete spectral analysis of the hybridized tissue, and hence extraction of the principal dye components used for the probe. This provides a means by which we can multiplex the barcoding probes using more spectral bandwidth and obtain gene expression profiling of up to 247 genes in a single nucleus. The statistical analysis of the expression of these genes in tissue combined with a preserved tissue morphology will provide an eventual platform for interrogation of patient specimens where the outcomes are known. The expression patterns obtained from tumor cells in pathological samples will allow a correlation of specific genes with disease progression.